Pyrogens are pyrogenetic substances which abnormally raise the body temperature of a homothermic animal in a very small amount. When a pyrogen is introduced into the human body, for example, by intravenous injection of a medicine contaminated with it, apart from the main activity of the medicine, the pyrogen causes severe fever. It is said that, when this action of pyrogen becomes serious, it causes severe fever accompanied with chill and shudder and, occasionally, death from a shock. Many substances such as bacterial substances, inflammatory substances, plant polysaccharides, blood group substances and the like have been known as pyrogens. Among them, bacterial substances have the most important influence on fever and are called as bacterial toxins. In general, bacterial toxins are classified into exotoxins and endotoxins. Particularly, it is said that an endotoxin acting as so-called O-antigen the main component of which is a cell wall lipopolysaccharide (LPS) of a gram negative bacterium has the strongest pyrogenicity.
Accordingly, it is very important to detect or determine a pyrogen content, for example, to prevent contamination of a pyrogen in the production of medicines.
As a method for detecting or determining a pyrogen content, for example, there has been hitherto known fever test using a rabbit, or Limulus test using a blood cell extract of Limulus polyphenus. Particularly, Limulus test has been often used from viewpoints of sensitivity, simplicity, quantitative properties and the like.
However, Limulus test is liable to be interfered or activated by various substances present during determination and, therefore, complicated pre-treatment is required or, sometimes, it becomes difficult to determine the pyrogen content depending upon a specimen. Further, there are some substances other than pyrogens which are positive in Limulus test and they interfere precise determination of the pyrogen content. In order to solve this problem, it has been proposed to use highly purified reagents. However, such reagents are very expensive. Further, it is considered to be difficult to determine a very small amount of pyrogens contained in a substance having a low solubility.
Recently, in order to detect an endotoxin which is one of pyrogens, there has been proposed a method which comprises bringing a pyrogen-free activated charcoal obtained by treatment with an acid into contact with a specimen and reacting the activated charcoal with Limulus lysate to carry out Limulus test easily and rapidly (Japanese Patent Laid Open Publication No. 152425/1981). However, this method is yet unsatisfactory from viewpoints of specificity, sensitivity and quantitative properties for pyrogens. Further, U.S. Pat. No. 4,491,660 discloses that a certain polymer which can adsorbs an endotoxin can be used for concentrating and detecting the endotoxin. However, this polymer is yet unsatisfactory from viewpoints of specificity, sensitivity and quantitative properties for pyrogens, too.
The present inventors have already found that pyrogens are specifically adsorbed by an adsorbent comprising a nitrogen-containing heterocyclic compound bonded to a water-insoluble carrier directly or through a spacer and has filed a patent application (Japanese Patent Laid Open Publication No. 183712/1982). Then, the present inventors have further studied and found that, by using such an adsorbent, a pyrogen content can be readily carried out by Limulus test in high specificity and sensitivity even in the presence of various interfering substances or other Limulus test positive substances.